Breast cancer, a genetically heterogeneous disease, is the most common malignancy in women. An estimation of approximately 800,000 new cases worldwide was reported each year (Parkin D M, et al., (1999). CA Cancer J Clin 49: 33-64). Mastectomy is still currently the first option for the medical treatment. Despite surgical removal of the primary tumors, relapse at local or distant sites may occur due to micrometastasis undetectable at the time of diagnosis (Saphner T, et al., (1996). J Clin Oncol, 14, 2738-46). Cytotoxic agents are usually administered as adjuvant therapy after surgery, aiming to kill those residual or pre-malignant cells. Treatment with conventional chemotherapeutic agents is often empirical and is mostly based on histological tumor parameters. In the absence of specific mechanistic understanding, target-directed drugs, are therefore becoming the bedrock treatment for breast cancer. Tamoxifen and aromatase inhibitors, two representatives of its kind, have proven to elicit efficacious responses when used as adjuvant or chemoprevention in patients with metastasized breast cancer (Fisher B, et al., (1998). J Natl Cancer Inst, 90, 1371-88; Cuzick J (2002). Lancet 360, 817-824). However, the drawback is that only patients' expressing estrogen receptors are sensitive to these drugs. Moreover, concerns have recently been raised regarding their side effects, for example endometrial cancer resulting from long term tamoxifen treatment and bone fractures resulting from aromatase therapy in the postmenopausal women (Coleman R E (2004). Oncology. 18 (5 Suppl 3), 16-20).
In spite of recent progress in diagnostic and therapeutic strategies, prognosis of patients with advanced cancers remains very poor. Although molecular studies have revealed the involvement of alterations in tumor suppressor genes and/or oncogenes in carcinogenesis, the precise mechanisms still remain to be elucidated.
cDNA microarray technologies have enabled the construction of comprehensive profiles of gene expression in normal and malignant cells, and the comparison of gene expression in malignant and corresponding normal cells (Okabe et al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61: 3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)). This approach facilitates the understanding of the complex nature of cancer cells, and helps to elucidate the mechanism of carcinogenesis. Identification of genes that are deregulated in tumors can lead to more precise and accurate diagnosis of individual cancers, and to the development of novel therapeutic targets (Bienz and Clevers, Cell 103:311-20 (2000)). To disclose mechanisms underlying tumors from a genome-wide point of view, and discover target molecules for diagnosis and development of novel therapeutic drugs, the present inventors analyzed the expression profiles of tumor cells using a cDNA microarray of 23,040 genes (Okabe et al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).
Studies designed to reveal mechanisms of carcinogenesis have already facilitated the identification of molecular targets for anti-tumor agents. For example, inhibitors of farnesyltransferase (FTIs), which were originally developed to inhibit the growth-signaling pathway related to Ras and whose activation depends on post-translational farnesylation, have been shown to be effective in treating Ras-dependent tumors in animal models (Sun J, et al., Oncogene. 1998; 16:1467-73). Clinical trials on humans, using a combination of anti-cancer drugs and the anti-HER2 monoclonal antibody, trastuzumab, to antagonize the proto-oncogene receptor HER2/neu, have achieved improved clinical responses and overall survival of breast cancer patients (Molina M A, et al., Cancer Res. 2001; 61:4744-9). A tyrosine kinase inhibitor, STI-571, which selectively inactivates bcr-abl fusion proteins, has been developed to treat chronic myelogenous leukemias wherein constitutive activation of bcr-abl tyrosine kinase plays a crucial role in the transformation of leukocytes. Agents of these kinds are designed to suppress oncogenic activity of specific gene products (O'Dwyer M E & Druker B J, Curr Opin Oncol. 2000; 12:594-7). Therefore, gene products commonly up-regulated in cancerous cells may serve as potential targets for developing novel anti-cancer agents.
For example, a new approach of cancer therapy using gene-specific siRNA was attempted in clinical trials (Bumcrot D et al., Nat Chem Biol 2006 December, 2(12): 711-9). RNAi seems to have already earned a place among the major technology platforms (Putral L N et al., Drug News Perspect 2006 July-August, 19(6): 317-24; Frantz S, Nat Rev Drug Discov 2006 July, 5(7): 528-9; Dykxhoorn D M et al., Gene Ther 2006 March, 13(6): 541-52). Nevertheless, there are several challenges that need to be faced before RNAi can be applied in clinical use. These challenges include poor stability of RNA in vivo (Hall A H et al., Nucleic Acids Res 2004 Nov. 15, 32(20): 5991-6000, Print 2004; Amarzguioui M et al., Nucleic Acids Res 2003 Jan. 15, 31(2): 589-95), toxicity as an agent (Frantz S, Nat Rev Drug Discov 2006 July, 5(7): 528-9), mode of delivery, the precise sequence of the siRNA or shRNA used, and cell type specificity. It is well-known fact that there are possible toxicities related to silencing of partially homologous genes or induction of universal gene suppression by activating the interferon response (Judge A D et al., Nat Biotechnol 2005 April, 23(4): 457-62, Epub 2005 Mar. 20; Jackson A L & Linsley P S, Trends Genet. 2004 November, 20(11): 521-4). Therefore, double-stranded molecules targeting cancer-specific genes devoid of adverse side-effects, are needed for the development of anticancer drugs.
Alternatively, it has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs) recognize epitope peptides derived from tumor-associated antigens (TAAs) presented on the MHC Class 1 molecule, and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological approaches (Boon, Int J Cancer 54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994)). Some of the discovered TAAs are now in the stage of clinical development as targets of immunotherapy. TAAs discovered to date include MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gp100 (Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al., J Exp Med 187: 277-88 (1998)), and NY-ESO-1 (Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997)). On the other hand, gene products which had been demonstrated to be specifically over-expressed in tumor cells, have been shown to be recognized as targets inducing cellular immune responses. Such gene products include p53 (Umano et al., Brit J Cancer 84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit J Cancer 84: 94-9 (2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)), and the like.
In spite of significant progress in basic and clinical research concerning TAAs (Rosenberg et al., Nature Med 4: 321-7 (1998); Mukherji et al., Proc Natl Acad Sci USA 92: 8078-82 (1995); Hu et al., Cancer Res 56: 2479-83 (1996)), only a limited number of candidate TAAs for the treatment of adenocarcinomas, including breast cancer, are currently available. TAAs abundantly expressed in cancer cells, and at the same time whose expression is restricted to cancer cells, would be promising candidates as immunotherapeutic targets. Further, identification of new TAAs inducing potent and specific anti-tumor immune responses is expected to encourage clinical use of peptide vaccination strategies in various types of cancer (Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994); Shichijo et al., J Exp Med 187: 277-88 (1998); Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Cancer Inst 88: 1442-55 (1996); Butterfield et al., Cancer Res 59: 3134-42 (1999); Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et al., J Immunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8 (1997); Fujie et al., Int J Cancer 80: 169-72 (1999); Kikuchi et al., Int J Cancer 81: 459-66 (1999); Oiso et al., Int J Cancer 81: 387-94 (1999)).
It has been repeatedly reported that peptide-stimulated peripheral blood mononuclear cells (PBMCs) from certain healthy donors produce significant levels of IFN-γ in response to the peptide, but rarely exert cytotoxicity against tumor cells in an HLA-A24 or -A0201 restricted manner in 51Cr-release assays (Kawano et al., Cancer Res 60: 3550-8 (2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et al., Jpn J Cancer Res 92: 762-7 (2001)). However, both of HLA-A24 and HLA-A0201 are popular HLA alleles in Japanese, as well as Caucasian populations (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al., J Immunol 155: 4307-12 (1995); Kubo et al., J Immunol 152: 3913-24 (1994); Imanishi et al., Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)). Thus, antigenic peptides of cancers presented by these HLAs may be especially useful for the treatment of cancers among Japanese and Caucasian populations. Further, it is known that the induction of low-affinity CTL in vitro usually results from the use of a peptide at a high concentration, generating a high level of specific peptide/MHC complexes on antigen presenting cells (APCs), which will effectively activate these CTL (Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7 (1996)).
To determine the mechanism of breast carcinogenesis and identify novel diagnostic markers and/or drug targets for the treatment of these tumors, the present inventors analyzed the expression profiles of genes in breast carcinogenesis using a genome-wide cDNA microarray containing 27,648 genes. From a pharmacological point of view, suppressing oncogenic signals is easier in practice than activating tumor-suppressive effects. Thus, the present inventors searched for genes that were up-regulated during breast carcinogenesis.
Since cytotoxic drugs often cause severe adverse reactions, thoughtful selection of novel target molecules on the basis of well-characterized mechanisms of action will facilitate development of effective anti-cancer drugs with minimum risk of side effects. Toward this goal, the inventors previously performed expression profile analysis of 81 breast cancers (Nishidate T et al., Int J Oncol 2004, 25: 797-819) and 29 normal human tissues (Saito-Hisaminato A et al., DNA Res 2002, 9: 35-45; WO05/028676) and found dozens of genes that were highly and universally up-regulated in breast cancer cells.
PBK (PDZ-binding kinase)/TOPK (T-LAK cell-originated protein kinase) gene is one of these genes which was found to be significantly over-expressed in the great majority of breast cancer cases examined (the PBK/TOPK gene is dubbed “A7870” in WO05/028676). Further, the present inventors demonstrated that a small-interfering RNA (siRNA) designed to reduce the expression of the PBK/TOPK gene has a growth-inhibitory effect on breast cancer cells expressing the gene.
PBK/TOPK is a member of the Ser/Thr kinase family and was first identified as a Dlg1-interacting protein by yeast two-hybrid screening and characterized as a mitotic kinase with PDZ-binding motif at the C-terminus (Gaudet S et al., Proc Natl Acad Sci USA 2000, 97: 5167-72). PBK/TOPK was also indicated by another group as a MAPKK-like protein kinase that phosphorylates p38 protein (Abe Y et al., J Biol Chem 2000, 275: 21525-31). In addition, the possible interaction between Raf and PBK/TOPK was shown by yeast two-hybrid screening analysis (Yuryev A et al., Genomics 2003, 81: 112-25). These two findings implied that PBK/TOPK might involve the MAPK pathway.
Post-translational modifications at the N-terminal portion of histone H3, including acetylation, methylation, and phosphorylation were described previously (Martin C & Zhang Y, Nat Rev Mol Cell Biol 2005, 6: 838-49; Nowak S J et al., Trends Genet. 2004, 214-20; Prigent C & Dimitrov S, J Cell Sci 2003, 116: 3677-85). Among them, phosphorylation of histone H3 at Ser10 is known to be involved in the initiation of mammalian chromosome condensation, an essential event in cell mitosis (Prigent C & Dimitrov S, J Cell Sci 2003, 116: 3677-85; Van Hooser A et al., J Cell Sci 1998, 111: 3497-506). According to the “ready production label” model, Ser10 phosphorylation of histone H3 reaches the maximum level in metaphase, as an indication that the chromosomes are ready to be separated, and then Ser10 is dephosphorylated accompanied by metaphase/anaphase transition (Hans F & Dimitrov S, Oncogene 2001, 20: 3021-7). Interestingly, previous reports indicated that okadaic acid (“OA”) induced Ser10 phosphorylation of histone H3 through inhibition of protein phosphatases (PPs) (Murnion M E et al., J Biol Chem 2001, 276: 26656-65; Eyers P A et al., Curr Biol 2003, 13: 691-7). For example, Aurora-A is known to be deactivated by protein phosphatase 2A (PP2A), but to be reactivated by its autophosphorylation through binding with TPX2 (Targeting protein for Xenopus kinesin-like protein 2) protein that impair the activity of PP2A (Eyers P A et al., Curr Biol 2003, 13: 691-7).
Entry into mitosis in mammalian cells is triggered by activation of the CDK1-cyclin B1 kinase targeting a lot of substrates to induce subsequent mitotic processes (Nigg E A., Nat Rev Mol Cell Biol 2: 21-32 (2001)). Those substrates are also involved in the late stage of cell mitosis through a phosphorylation by CDK1-cyclin B1 complex; APC (anaphase-promoting complex) ubiqutin ligase that is activated to initiate mitotic exit (Kraft C et al., EMBO J. 22: 6598-609 (2003)) and conformational proteins that obtain a docking site with PLK1, such as INCENP (inner centromere protein, Goto H et al., Nat Cell Biol 8: 180-7 (2006)) and PRC1 (protein regulator of cytokinesis 1, Neef R et al., Nat Cell Biol 9: 436-44 (2007)) required for metaphase-anaphase transition and cytokinesis, respectively. Moreover, it implies a role of close cooperation between protein kinases and phosphatases to promote cell mitosis because recent works reported that the activity of Protein phosphatase 1 (PP1α has an inactive phosphorylation site (Thr320) targeted by CDK1-cyclin B1 kinase (Kwon Y G et al., Proc Natl Acad Sci USA 94: 2168-73 (1997)). Although it has been reported that PBK/TOPK can be phosphorylated at Thr9 by CDK1-cyclin B1, how activation of PBK/TOPK by CDK1-cyclin B1 complex mitotic cells and its function of in cell proliferation and cancer progression is still largely unknown.